1. Field of the Invention
The present invention relates to an apparatus and system for DNA detection such as detection of single nucleotide mutations, gene diagnosis, genetic typing or detecting biological substances such as protein and ATP.
2. Description of the Related Art
Analysis of the human genome sequence is almost completed, and activity is increasing in efforts to put genetic information to practical use in the medical field in areas such as diagnosis. Following the analysis of the genome sequence, gene expression profile analysis and analysis of single nucleotide polymorphisms (SNPs) in genes are now attracting attention. By examining genes expressing under a variety of conditions and investigating gene mutations of a variety of solid bodies, genetic functions and the relation between genes and disease or genes and sensitivity to pharmaceuticals can be investigated. Further, the diagnosis of disease and the like is now being carried out using such accumulated knowledge about genes.
Unlike analysis of unknown genes, in diagnosis of disease the object of investigation is a known gene or the presence or absence of a mutation thereof. It is desirable that such investigation can be performed at a low cost, and various methods have been developed to achieve this. In medical diagnosis, examination of disease occurring due to the influence of a variety of genes and environments as well as examination of genes related to sensitivity to pharmaceuticals is becoming more important than diagnosis of disease caused by a single gene. To achieve this, it is important to simultaneously analysis a variety of types of genes. Accordingly, it is necessary to examine a plurality of genes, and not just a single gene or mutation, and a method is thus required that determines SNPs and the like at a low cost that includes the amplification process of an assay site of the gene. Methods reported as practicable in analysis of SNPs and probe assay of genes include Invader assay (Science 260, 778 (1993)), TaqMan assay (J. Clin. Microbiol. 34, 2933 (1996)), DNA microarrays (Nature Gent. 18, 91 (1998), pyrosequencing (Science 281, 363 (1998)) and the like. The first 3 methods mentioned are detection methods that use fluorescence labeling, and employ an excitation laser light source and a light sensor system. In contrast, pyrosequencing is a method that uses stepwise complementary strand synthesis and chemiluminescence, and employs a system that sequentially injects trace amounts of nucleic acid substrate and a light sensor system, and does not require an excitation light source.
FIG. 1 shows an example of a configuration of an apparatus for measuring chemiluminescence used in a conventional method. As a detector 260 used as a mechanism for detecting luminescence from a plurality of reaction baths 101 on a sample plate 100, for example, a photomultiplier tube is used. From a container 133 containing sample DNA, a sample is dispensed to each of the reaction baths 101 on sample plate 100 by means of a pipette 135. Next, a reagent solution containing primers corresponding to a plurality of measurement items is dispensed from a container 131 by means of a pipette 134. To prevent contamination with a different reagent, pipette 134 is washed in lavage fluid of a container 132. To detect luminescence induced in the reaction baths in accordance with matching between a reagent and sample, detector 260 scans the reaction baths by means of a moving device 136. Because a device to move the detector or the sample plate is indispensable in this apparatus, it is difficult to implement miniaturization, cost reduction and increased throughput.
As a light-sensitive detector to measure faint light, in general, a photomultiplier tube, charge-coupled device (CCD) or MOS-type sensor or the like is mainly used. While a significant advantage can be obtained by the use of a photomultiplier tube, it requires a high voltage, and integration is also a problem, and thus it is not suitable for a small-sized apparatus. In contrast, large-scale integration is possible with a semiconductor sensor, and it also operates on a low voltage and low current and is therefore suitable for miniaturization of an apparatus. Apart from the case of an avalanche photodiode, which is not suitable for integration as the production process thereof is complicated, for a semiconductor sensor, the quantum efficiency of a commonly used CCD or photodiode is at highest 1, which is low compared to the photomultiplier tube. To enhance the S/N ratio a strategy that effectively utilizes chemiluminescence is required.
In measuring luminescence from a biological sample, it is necessary to amplify only the signal of interest under a measurement condition of a high background light intensity. Further, in the case of measurement by a charge storage technique using a photodiode as a detector, because the potential decrement from a charging potential of about 1–5 V becomes the signal output, to perform a large amplification for a micro-signal, a mechanism to cancel the charging potential is necessary to prevent saturation of the amplifier. Due to such necessity, a technique is used which measures a reference signal along with the signal from the sample of interest, and enhances the S/N ratio by conducting differential amplification of these signals. At this time, a method can be employed in which a specific location on the sample plate is defined as a control pixel, and which then determines the differential amplification of the signal from the sample of interest using the control pixel as a basis. Alternatively, a method can be employed in which a signal is read in a condition where light is not irradiated to the sensor, this signal is then stored in a temporary capacitor for record, and then a signal light is irradiated and the differential amplification between that signal and the previously stored signal is determined (IEEE Transactions on electron devices vol. 41, 452 (1994)). In the former method, there is no flexibility in the selection of a control pixel and there is also difficulty in terms of usability in measuring relative luminescence intensity from a plurality of different samples. In the latter method, there is difficulty involved in the constitution of a capacitor that can retain an electric charge even for a signal accumulated over several seconds, and also in obtaining high measurement accuracy that excludes the influence of dark current fluctuations. Therefore, there is a need for an advanced correction method that solves these problems.
The realization of measurement with high throughput is essential to prepare for greater utilization of DNA diagnosis, such as that concerning SNPs. However, while simultaneous measurement of a plurality of items is effective, in measurement that utilizes chemiluminescence, conventionally, for each reaction bath on a sample plate, reaction of one type of sample and probe has been performed, and it has been difficult to obtain high throughput for a large number of assay items or a large number of samples.